Modified glutamic acid decarboxylase (GAD)

ABSTRACT

A modified GAD especially characterized by a substantially preserved immunogenicity in connection with diabetes compared with a non-modified GAD and a reduced or non-existent effectivity in connection with a GABA-synthesis. The invention relates also to a method for manufacturing a modified GAD, a method for supplying a modified GAD, nucleotide sequence encoding the modified GAD, a vector containing the nucleic acid sequence, a host, a pharmaceutical composition comprising modified GAD and use thereof for treating and/or preventing autoimmune disorders such as IDDM.

The present invention relates to a genetically-modified recombinant formof the 65 kDa human glutamic acid decarboxylase (hGAD65) that retainsstructural integrity for immuno-reactivity characteristic of theunmodified form, but that is without (or with considerably reduced)enzyme activity. The novel modified GAD is advantageously used inpharmaceutical compositions for treating autoimmune diseases.

BACKGROUND OF THE INVENTION

The inhibitory neurotransmitter γ-aminobutyric acid (GABA), derived fromL-glutamic acid by GAD is present in brain as well as several tissuesoutside the central nervous system. Biological functions of GAD and GABAextend beyond regulation of neurotransmission to include effects on theimmune system as well as modulation of cell proliferation, proteinsynthesis and metabolism.

GAD has recently been associated with autoimmune insulin-dependentdiabetes mellitus (IDDM) (Boekkeskov et al, Nature 347:151 (1990),incorporated by reference) because of the increased incidence of IDDM inpatients with stiff-man syndrome, a disease associated withautoantibodies against GAD. Antibodies from diabetic sera have also beenshown to bind GAD. At least two different GAD, i.e. GAD 65 and 67 havebeen identified and sequenced in human beings. hGAD65 and hGAD67 haveseveral epitopes in common. However, different epitopes are involved indifferent reactions and contexts.

Binding of the coenzyme pyridoxal-5'-phospate (PLP) to GAD was firstreported by Roberts & Frankel in 1951 (J. Biol. Chem. 188:789, fullyincorporated in the description by reference), and formation of a Schiffbase during GAD-mediated decarboxylation was subsequently proposed by WT Jenkins 1961, Fed. Proc. 20:978 (fully incorporated in the descriptionby reference) (i.e. referenced in Roberts & Simonsen 1963, Biochem.Pharmacol. 12:113-134, fully incorporated in the description byreference). Accordingly, PLP has been identified as a critical co-factorin decarboxylation by GAD.

Cloning of GAD is disclosed in WO92/05446 and WO92/20811 (bothincorporated by reference).

Location of the PLP binding site within the amino acid sequence ofhGAD65 was enabled by publication of the cDNA sequence of this enzyme(P.N.A.S. 88:8337, fully incorporated in the description by reference).The site was found because of the homology centred around the amino acidlysine at a/a #396 in hGAD65--to the sequence: X-His-Lys-X that waspreviously published as the PLP binding site in crystallised E.coli GAD(Biochemistry 9:226-233; Biochemistry 13:670-676, both of which arefully incorporated in the description by reference).

This region in hGAD65 has the following amino acid sequence (SEQ IDNOS:9 and 10):

    ______________________________________                                        Thr  Trp    Asn    Pro  His  Lys* Met  Met  Gly  Val                          T    W      N      P    H    K*   M    M    G    V                            •                      •             •                      #391                         #396                #400                         ______________________________________                                         *selected herein for mutation to change K to Arg (R)                     

This homology in this region has subsequently been confirmed by Lernmark(unpublished) using computer homology matches to ca. 10 otherPLP-binding (non-GAD) proteins.

The approach of the present invention is a novel GAD without enzymeactivity by mutating a single codon in the PLP binding site in the cDNAsequence for hGAD65. This results in the substitution of a single aminoacid that is incapable of supporting wild-type enzyme activity.

As the lysine at #396 has been identified as critical for enzymeactivity (via formation of a Schiff base during GAD-mediateddecarboxylation), the present invention is directed to substitute anamino acid incapable of Schiff base formation at position #396 as asubtle means of achieving this effect.

In addition, a "conservative amino acid substitution" (i.e. replacementof lysine by an amino acid of comparable size and hydrophobicity) isintended to minimise any structural or conformational changes to thehGAD65 protein, and thereby avoid alterations to either antigenicetitopes or T cell determinants originally present in recombinant humanGAD (rhGAD65).

SUMMARY OF THE INVENTION

The present invention relates to a modified, non-enzymically activehGAD65 retaining the immunoreactive characteristics of wild type hGAD65,wherein amino acid #396 has been altered an L-amino acid other thanlysine (SEQ ID NO:3). It is preferred to alter amino acid #396 to anamino acid chosen from the group of isoleucine (SEQ ID NO:4), arginine(SEQ ID NO:5), glutamine (SEQ ID NO:6), histidine (SEQ ID NO:7) orglycine (SEQ ID NO:8). In a particularly preferred embodiment of thepresent invention amino acid #396 is altered to arginine.

DETAILED DESCRIPTION OF THE INVENTION

As already mentioned, the modified hGAD65 protein having considerablyreduced enzymatic activity constitutes an object of the presentinvention. This altered protein can be used for treating auto-immunediseases, such as IDDM.

"Modified hGAD65 protein" as utilized herein refers to human glutamicacid decarboxylase having a molecular weight of 65 kDa wherein aminoacid #396 has been altered to an amino acid other than lysine, and whoseenzymatic activity has been considerably reduced. In a preferredembodiment, the altered amino acid is chosen from the group ofisoleucine, arginine, glutamine, histidine and glycine. Of these,arginine is considered to be most preferred as it is unable to form aSchiff base, but has similar hydrophobicity (index of both is 3.0) andsize compared to lysine. The amino acid sequence of this modified hGAD65protein is disclosed as SEQ ID NO:5.

The present invention also relates to nucleic acid sequences encoding amodified hGAD65 protein taken into account the degenerated genetic code.A preferred embodiment is disclosed as SEQ ID NO:2.

In an other aspect, the present invention relates to an oligonucleotidethat can be used for altering amino acid #396 to an amino acid otherthan lysine by site-directed mutagenesis of a nucleic acid encodingwild-type hGAD65. A preferred embodiment of such an oligonucleotide isdisclosed as SEQ ID NO:1.

In another aspect, the present invention relates to vectors and hostcells comprising the above mentioned nucleic acids encoding modifiedhGAD65 protein. The nucleic acids may be readily introduced into a widevariety of host cells. Representative examples of such host cellsinclude plant cells, eukaryotic cells and prokaryotic cells. Withinpreferred embodiments, the nucleic acid molecules are introduced intocells from an insect or a vertebrate or warm-blooded anomal, such as ahuman, macaque, dog, cow, horse, pig, sheep, rat hamster, mouse, or afish, or any hybrid thereof.

The nucleic acid molecules (or vectors) may be introduced into hostcells by a wide variety of mechanisms, including for example calciumphosphate-mediated transfection (Wigler et al., Cell 14:725, 1978, fullyincorporated in the description by reference), lipofection; gene gun(Corsaro and Pearson, Somatic Cell Gen. 1:603, 1981; Graham and Van derEb, Virology 52:456, 1973; both of which are fully incorporated in thedescription by reference), electroporation (Neumann et al., EMBO J.1:841-845, 1982, fully incorporated in the description by reference),retroviral, adenoviral protoplast-mediated transfection or DEAE-dextranmediated transfection (Ausubel et al., (eds), Current Protocols inMolecular Biology, John Wiley and Sons, Inc., NY, N.Y., USA 1987, fullyincorporated in the description by reference) as well as baculovirustransfection, such as the method described in EP 0 327 180 (fullyincorporated in the description by reference).

As noted above, the present invention also provides a variety ofpharmaceutical compositions for treating and/or preventing autoimmunediseases, such as IDDM, comprising pharmaceutically active amount of amodified hGAD65 protein along with a pharmaceutically or physiologicallyacceptible carrier, excipients or diluents. Generally, such carriersshould be non-toxic to recipients at the dosages and concentrationsemployed. Ordinarily, the preparation of such compositions entailscombining the therapeutic agent with buffers, antioxidants such asascorbic acid, low molecular weight (less than about 10 residues)polypeptides, proteins, amino acids, carbohydrates including glucose,sucrose or dextrins, chelating agents such as EDTA, glutathione andother stabilizers and excipients. Neutral buffered saline or salinemixed with non-specific serum albumin are exemplary appropriatediluents.

In addition, the pharmaceutical compositions of the present inventionmay be prepared for administration by a variety of routes, including forexample orally, sublingually, rectally, intraarticularly,intracranially, intradermally, intramuscularly, intraocularly,intraperitoneally, intravenously or subcutaneously. The compositions canoccur as tablets, sublingual tablets, granules, pills, capsules,dragees, solutions, syrups, mixtures, emulsions, suppositorys, aerosols,etc.

Furthermore, the above mentioned nucleic acid molecules can be used aspharmaceuticals and directly be injected into a human or animal in needof such treatment by using a gene gun (Corsaro and Pearson, Somatic CellGen. 1:603, 1981; Graham and Van der Eb, Virology 52:456, 1973; both ofwhich are fully incorporated in the description by reference). Thetreated human or animal body are then able to synthesize the modifiedhGAD65 according to the present invention.

As already mentioned, GAD is expressed by CNS, but also by islet cellsin pancrease even producing insulin. GAD 65 is regarded to be theantigen initiating the immune response leading to diabetes, type 1(IDDM). A GAD similar antigen has also been identified on Epstein Barrtransformed lymphocytes and in coxsaclievirus-infected cells. Theinfluence of GAD 65 and the relation between IDDM and the presence ofGAD 65 are described in Kaufman et al., Nature 361:69, 1993; and Nov.4th, 1993, pp 69-71, incorporated by reference.

Accordingly the present invention concerns a method to prevent and totreat IDDM by inducing a tolerance to GAD as well as a GAD for such atreatment. The method can be used on all or certain population segmentswith the object to eliminate or to minimize the IDDM frequency, at leastof GAD initiated IDDM. The method can be adapted above all toindividuals with a genetic predisposition for IDDM and to individualswith an increased antibody titer against GAD with the object to lead theat least in the last mentioned case already started autoimmune reactionin regress. The method can also be used with patients with a developedIDDM to eliminate the possibility of a renewned autoimmune attack aftere.g. a pancreas or an islet cell transplantation. The method relates tromodifying the DNA sequence coding GAD in such a way that the efficiencyof the produced peptide in the connection with GABA decreases orvanishes without thereby its diabetes autoimmune antigenic featuresdisappearing. In this way GAD can be used more safely for human beingsand thus GAD can be administered in various ways for introducingtolerance.

For the participation of GAD as an enzyme during the splitting ofGlutamatic Acid into GABA and carbon dioxide the presence of a cofactorPLP (pyridoxal-5'-phosphate) is required. The PLP binding site consistsof aminoacid 395-397 of wild type hGAD65. By replacing the aminoacid#396 with another amino acid the binding site for PLP is influenced sothat said cofactor PLP is not bound to said binding site on the GADmolecule resulting in that said GAD molecule no longer participates inthe GABA synthesis. The modification at said binding site is notinfluencing the GAD epitope being diabetogenerating. The modified GADmolecule is expressed with a known recombinant technique.

The manufacturing of cDNA from mRNA is well known today. The modifiedhGAD65 protein produced is then used to treat IDDM.

This modified hGAD65 so obtained is useful as an immunomodulanttherapeutic to prevent certain autoimmune diseases (includinginsulin-dependent diabetes mellitus) and certain other diseases(including neurological diseases). The provision of this is a novelmeans to avoid (or minimise) the risk of toxicity (e.g. neurotoxicity)possibly arising from the enzyme activity of a GAD-basedimmuno-therapeutic.

From now on, the invention will be described with reference to theenclosed examples. However, it should be understood that the scope ofthe present invention is not to be restricted to those examples.

Materials and methods

1. DNA oligomer design and synthesis

Both oligos (below) represent sequences from the coding strand of boththe insert or vector, and are intended to hybridise to the same (ie.non-coding) strand of p65 (Synectics proprietary clone derived from thecDNA sequence for human GAD65 published by Karlsen et al 1991 Proc.Natl. Acad. Sci USA 88:8337-8341 and Bu et al. 1992 Proc. Natl. Acad.Sci USA 89:2115-2119; both of which are fully incorporated in thedescription by reference).

The mutagenic primer (SEQ ID NO:1) was designed according to Stratagenerecommendations and contained the CGC codon for Arginine, flanked ateither end by 14 nts derived from the corresponding hGAD65 cDNAsequence. Of the 6 alternative codons for Arginine (viz. AGA, AGG, CGA,CGC, CGG, CGU), the codon CGC was selected as this has the greatestusage in animals (Grantham R, Gautier C, Gouy M, Jacobzone M, Mercier R1981 Nucleic Acid Res 9:43-74: fully incorporated in the description byreference). Accordingly, the 31-mer mutagenic oligo has sequence:

5'-CG TGG AAT CCA CAC CGC ATG ATG GGA GTC CC-3' (SEQ ID NO:1)

-corresponding to nt #1186 (upstream end) and #1216 (downstream) of p65.This mutagenic primer (coded "SDM-PLP") was synthesised at theKaronlinska Institute facility, and kinased (via T4 polynycleotidekinase, see Current Protocols in Molecular Biology, John Wiley & Sons,Inc., fully incorporated in the description by reference) prior to use.The Tm of SDM-PLP is estimated as 60° C. and was used duringmutagenesis.

The Sca I to Mlu I selection primer was chosen because of the absence ofsites in the rhGAD65 insert and the single site in the pGEM4z vector ofp65. This kinased primer was supplied by Stratagene (catalogue #300331).

2. Site-directed mutagenesis of proprietary hGAD65 clone "p65"

The Chameleon™ double-stranded DNA site-directed mutagenesis kit fromStratagene was used for modification of the Synectics BiotechnologyrhGAD65 clone (coded "p65"), and followed the manufacturers'instructions in the instruction manual (Catalog #200509, Jun. 13, 1994,fully incorporated in the description by reference).

3. DNA sequencing of candidate clones

The required mutation (present in clone #18) was confirmed by DNAsequencing using "Taq DyeDeoxy Terminator Cycle Sequencing" (AppliedBioSystems) and "ALF" sequenator at the Karonlinska facility. Thesequencing oligo "JR11" (annealing to nts #1353-1369 in the codingstrand) was used to confirm sequence at nts "1200-1202. Oligo "JR4"(annealing to the noncoding strand upstream of nts "1200-1202, at nts#1033-1050) was used to obtain seqence in a downstream direction to thesite of the mutation.

Methods used for these manipulations followed previously-publishedprocedures (e.g. Current Protocols in Molecular Biology, John Wiley &Sons, Inc; Sambrook, Fritsch & Maniatis Molecular Cloning: A LaboratoryManual. Cold Spring Harbor Laboratory Press, 1989; both of which arefully incorporated in the description by reference).

4. Check for comparable immunoreactivity between mutant and wild-type

Immunoreactivity of the in vitro transcribed/translated mutant rhGAD65cDNA (clone #18) was shown to be indistinguishable from the original(non-mutant) p65 clone by using IDDM sera in a radioimmunoassay specificfor GAD65 autoantibodies (Falorni et al. 1993 Diabetologia 36 (suppl. 1)A45; Falorni et al. 1994 Autoimmunity 19:113-125; both of which arefully incorporated in the description by reference).

This analysis compared immunoreactivity of the mutant and wild-typeclones by comparing radioactivity precipitated by ca. 20GAD65-positiveIDDM sera on incubation with ³⁵ S-labelled rhGAD65 protein synthesisedin vitro from either the wild-type or mutant rhGAD65 cDNA clones.

EXAMPLE 1 Site-directed Mutagenesis of Wild-type hGAD65

An oligonucleotide having the sequence:

5'-CG TGG AAT CCA CAC CGC ATG ATG GGA GTC CC-3' (SEQ ID NO:1)

was synthesized, phosphorylated and used to mutate a cDNA having thesequence of FIG. 2 in Karlsen et al., P.N.A.S. 88:8337, 1991(incorporated into the description by reference) according theinstructions in the above mentioned Chamelon™ kit. Competent Escherichiacoli F'11 rec A cells were transformed with p65 mutant plasmids.Positive clones were screened by DNA sequencing using the Taq DyeDeoxy™Terminator Cycle Sequencing Kit from Applied Biosystems. A clonecontaining the desired mutatant were found.

EXAMPLE 2 In Vitro Translation of ³⁵ S-mutant hGAD65

Recombinant modified human GAD65 was produced by in vitrotranscription/translation of the cDNA inserted into the pGEM4z vector.The translation reaction was carried out using SP6 RNA polymerase andrabbit reticulocyte lysate. The materials used in the experiment werepart of the TNT coupled transcription/translation system from Promega.

The following reaction mixture was prepared:

Plasmid DNA encoding modified hGAD65 (2 mg/ml) 1 μl

RNAsin (Promega) 1 μl

TNT buffer 2 μl

Amino acid mixture (without methionine)(Promega) 1 μl

³⁵ S-methionine (NEN-Dupont) 4 μl

SP6 RNA polymerase (Promega) 1 μl

Rabbit Reticulocyte lysate (Promega) 25 μl

Water up to 50 μl

The reactions were incubated for 90 minutes at 30° C. At the end of theincubation time, 2 ml from the reaction tube was incubated for 10minutes at 37° C. with 1M NaOH/2% H₂ O₂ and 30 minutes on ice with 0.9ml 25% trichloroacetic acid (TCA) in order to precipitate proteins thatmay have been formed. The TCA precipitate was collected on a GF-AWhatman glass fibre filter and its radioactivity was evaluated in aliquid scintillation analyser. It was shown that the precipitate wasradioactive and hence, modified hGAD65 was formed.

EXAMPLE 3 Immunoprecipitation of 35S-modified hGAD65 with IDDM Sera

In vitro translated modified hGAD65 from example 2 wasimmunoprecipitated with IDDM sera to test the immunoreactivity. For eachserum sample 15000 cpm TCA-precipitable GAD65 was precipitated with 2 μlof human serum from healthy or diabetic individuals (final serumdilution 1:50). After overnight immunoprecipitation in cold room,antibody bound ³⁵ S-modified hGAD65 was separated from free ³⁵S-modified hGAD65 by protein A Sepharose using a Multiscreen AssayMillipore system. Immunoprecipitated radioactivity was evaluated in aliquid scintillation analyzer.

Immunoprecipitation of ³⁵ S-modified hGAD65 for diabetic sera wassignificantly higher than that obtained with sera from healthy persons.The used diabetic sera were previously found positive for antibodiesagainst wild type hGAD65. Hence, it was concluded that modified hGAD65has an immunoreactivity similar to that of wild type hGAD65.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - <160> NUMBER OF SEQ ID NOS: 14                                              - <210> SEQ ID NO 1                                                           <211> LENGTH: 31                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:R INFORMATION: Description of Artificial                            #No. 396 of hGAD65ide for altering residue                                    - <400> SEQUENCE: 1                                                           #          31      catg atgggagtcc c                                          - <210> SEQ ID NO 2                                                           <211> LENGTH: 1761                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:ArtificialION: Description of Artificial                                  gene encoding mutant hGAD65                                             - <400> SEQUENCE: 2                                                           - atggcatctc cgggctctgg cttttggtct ttcgggtcgg aagatggctc tg - #gggattcc         60                                                                          - gagaatcccg gcacagcgcg agcctggtgc caagtggctc agaagttcac gg - #gcggcatc        120                                                                          - ggaaacaaac tgtgcgccct gctctacgga gacgccgaga agccggcgga ga - #gcggcggg        180                                                                          - agccaacccc cgcgggccgc cgcccggaag gccgcctgcg cctgcgacca ga - #agccctgc        240                                                                          - agctgctcca aagtggatgt caactacgcg tttctccatg caacagacct gc - #tgccggcg        300                                                                          - tgtgatggag aaaggcccac tttggcgttt ctgcaagatg ttatgaacat tt - #tacttcag        360                                                                          - tatgtggtga aaagtttcga tagatcaacc aaagtgattg atttccatta tc - #ctaatgag        420                                                                          - cttctccaag aatataattg ggaattggca gaccaaccac aaaatttgga gg - #aaattttg        480                                                                          - atgcattgcc aaacaactct aaaatatgca attaaaacag ggcatcctag at - #acttcaat        540                                                                          - caactttcta ctggtttgga tatggttgga ttagcagcag actggctgac at - #caacagca        600                                                                          - aatactaaca tgttcaccta tgaaattgct ccagtatttg tgcttttgga at - #atgtcaca        660                                                                          - ctaaagaaaa tgagagaaat cattggctgg ccagggggct ctggccatgg ga - #tattttct        720                                                                          - cccggtggcg ccatatctaa catgtatgcc atgatgatcg cagcgtttaa ga - #tgttccca        780                                                                          - gaagtcaagg agaaaggaat ggctgctctt cccaggctca ttgccttcac gt - #ctgaacat        840                                                                          - agtcattttt ctctcaagaa gggagctgca gccgtaggga ttggaacaga ca - #gcgtgatt        900                                                                          - ctgattaaat gtgatgagag agggaaaatg attccatctg atcttgaaag aa - #ggattctt        960                                                                          - gaagccaaac agaaagggtt tgttcctttc ctcgtgagtg ccacagctgg aa - #ccaccgtg       1020                                                                          - tacggagcat ttgaccccct cttagctgtc gctgacattt gcaaaaagta ta - #agatctgg       1080                                                                          - atgcatgtgg atgcagcttg gggtggggga ttactgatgt cccgaaaaca ca - #agtggaaa       1140                                                                          - ctgagtggcg tggagagggc caactctgtg acgtggaatc cacaccgcat ga - #tgggagtc       1200                                                                          - cctttgcagt gctctgctct cctggttaga gaagagggat tgatgcagaa tt - #gcaaccaa       1260                                                                          - atgcatgcct cctacctctt tcagcaagat aaacattatg acctgtccta tg - #acactgga       1320                                                                          - gacaaggcct tacagtgcgg acgccacgtt gatgttttta aactatggct ga - #tgtggagg       1380                                                                          - gcaaagggga ctaccgggtt tgaagcgcat gttgataaat gtttggagtt gg - #cagagtat       1440                                                                          - ttatacaaca tcataaaaaa ccgagaagga tatgagatgg tgtttgatgg ga - #agcctcag       1500                                                                          - cacacaaatg tctgcttctg gtacattcct ccaagcttgc gtactctgga ag - #acaatgaa       1560                                                                          - gagagaatga gtcgcctctc gaaggtggct ccagtgatta aagccagaat ga - #tggagtat       1620                                                                          - ggaaccacaa tggtcagcta ccaacccttg ggagacaagg tcaatttctt cc - #gcatggtc       1680                                                                          - atctcaaacc cagcggcaac tcaccaagac attgacttcc tgattgaaga aa - #tagaacgc       1740                                                                          #                1761ta a                                                     - <210> SEQ ID NO 3                                                           <211> LENGTH: 585                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:Modified,TION: Description of Artificial                                  non-enzymically active hGAD65 retaining - # the                         #type hGAD65.eactive characteristics of wild                                        Xaa at position 396 is Ile, Ar - #g, Gln, His or Gly.                   - <400> SEQUENCE: 3                                                           - Met Ala Ser Pro Gly Ser Gly Phe Trp Ser Ph - #e Gly Ser Glu Asp Gly         #                 15                                                          - Ser Gly Asp Ser Glu Asn Pro Gly Thr Ala Ar - #g Ala Trp Cys Gln Val         #             30                                                              - Ala Gln Lys Phe Thr Gly Gly Ile Gly Asn Ly - #s Leu Cys Ala Leu Leu         #         45                                                                  - Tyr Gly Asp Ala Glu Lys Pro Ala Glu Ser Gl - #y Gly Ser Gln Pro Pro         #     60                                                                      - Arg Ala Ala Ala Arg Lys Ala Ala Cys Ala Cy - #s Asp Gln Lys Pro Cys         # 80                                                                          - Ser Cys Ser Lys Val Asp Val Asn Tyr Ala Ph - #e Leu His Ala Thr Asp         #                 95                                                          - Leu Leu Pro Ala Cys Asp Gly Glu Arg Pro Th - #r Leu Ala Phe Leu Gln         #           110                                                               - Asp Val Met Asn Ile Leu Leu Gln Tyr Val Va - #l Lys Ser Phe Asp Arg         #       125                                                                   - Ser Thr Lys Val Ile Asp Phe His Tyr Pro As - #n Glu Leu Leu Gln Glu         #   140                                                                       - Tyr Asn Trp Glu Leu Ala Asp Gln Pro Gln As - #n Leu Glu Glu Ile Leu         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Met His Cys Gln Thr Thr Leu Lys Tyr Ala Il - #e Lys Thr Gly His Pro         #               175                                                           - Arg Tyr Phe Asn Gln Leu Ser Thr Gly Leu As - #p Met Val Gly Leu Ala         #           190                                                               - Ala Asp Trp Leu Thr Ser Thr Ala Asn Thr As - #n Met Phe Thr Tyr Glu         #       205                                                                   - Ile Ala Pro Val Phe Val Leu Leu Glu Tyr Va - #l Thr Leu Lys Lys Met         #   220                                                                       - Arg Glu Ile Ile Gly Trp Pro Gly Gly Ser Gl - #y Asp Gly Ile Phe Ser         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Pro Gly Gly Ala Ile Ser Asn Met Tyr Ala Me - #t Met Ile Ala Arg Phe         #               255                                                           - Lys Met Phe Pro Glu Val Lys Glu Lys Gly Me - #t Ala Ala Leu Pro Arg         #           270                                                               - Leu Ile Ala Phe Thr Ser Glu His Ser His Ph - #e Ser Leu Lys Lys Gly         #       285                                                                   - Ala Ala Ala Leu Gly Ile Gly Thr Asp Ser Va - #l Ile Leu Ile Lys Cys         #   300                                                                       - Asp Glu Arg Gly Lys Met Ile Pro Ser Asp Le - #u Glu Arg Arg Ile Leu         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Glu Ala Lys Gln Lys Gly Phe Val Pro Phe Le - #u Val Ser Ala Thr Ala         #               335                                                           - Gly Thr Thr Val Tyr Gly Ala Phe Asp Pro Le - #u Leu Ala Val Ala Asp         #           350                                                               - Ile Cys Lys Lys Tyr Lys Ile Trp Met His Va - #l Asp Ala Ala Trp Gly         #       365                                                                   - Gly Gly Leu Leu Met Ser Arg Lys His Lys Tr - #p Lys Leu Ser Gly Val         #   380                                                                       - Glu Arg Ala Asn Ser Val Thr Trp Asn Pro Hi - #s Xaa Met Met Gly Val         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Pro Leu Gln Cys Ser Ala Leu Leu Val Arg Gl - #u Glu Gly Leu Met Gln         #               415                                                           - Asn Cys Asn Gln Met His Ala Ser Tyr Leu Ph - #e Gln Gln Asp Lys His         #           430                                                               - Tyr Asp Leu Ser Tyr Asp Thr Gly Asp Lys Al - #a Leu Gln Cys Gly Arg         #       445                                                                   - His Val Asp Val Phe Lys Leu Trp Leu Met Tr - #p Arg Ala Lys Gly Thr         #   460                                                                       - Thr Gly Phe Glu Ala His Val Asp Lys Cys Le - #u Glu Leu Ala Glu Tyr         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Leu Tyr Asn Ile Ile Lys Asn Arg Glu Gly Ty - #r Glu Met Val Phe Asp         #               495                                                           - Gly Lys Pro Gln His Thr Asn Val Cys Phe Tr - #p Tyr Ile Pro Pro Ser         #           510                                                               - Leu Arg Thr Leu Glu Asp Asn Glu Glu Arg Me - #t Ser Arg Leu Ser Lys         #       525                                                                   - Val Ala Pro Val Ile Lys Ala Arg Met Met Gl - #u Tyr Gly Thr Thr Met         #   540                                                                       - Val Ser Tyr Gln Pro Leu Gly Asp Lys Val As - #n Phe Phe Arg Met Val         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Ile Ser Asn Pro Ala Ala Thr His Gln Asp Il - #e Asp Phe Leu Ile Glu         #               575                                                           - Glu Ile Glu Arg Leu Gly Gln Asp Leu                                         #           585                                                               - <210> SEQ ID NO 4                                                           <211> LENGTH: 585                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:ModifiedATION: Description of Artificial                                  hGAD                                                                    - <400> SEQUENCE: 4                                                           - Met Ala Ser Pro Gly Ser Gly Phe Trp Ser Ph - #e Gly Ser Glu Asp Gly         #                 15                                                          - Ser Gly Asp Ser Glu Asn Pro Gly Thr Ala Ar - #g Ala Trp Cys Gln Val         #             30                                                              - Ala Gln Lys Phe Thr Gly Gly Ile Gly Asn Ly - #s Leu Cys Ala Leu Leu         #         45                                                                  - Tyr Gly Asp Ala Glu Lys Pro Ala Glu Ser Gl - #y Gly Ser Gln Pro Pro         #     60                                                                      - Arg Ala Ala Ala Arg Lys Ala Ala Cys Ala Cy - #s Asp Gln Lys Pro Cys         # 80                                                                          - Ser Cys Ser Lys Val Asp Val Asn Tyr Ala Ph - #e Leu His Ala Thr Asp         #                 95                                                          - Leu Leu Pro Ala Cys Asp Gly Glu Arg Pro Th - #r Leu Ala Phe Leu Gln         #           110                                                               - Asp Val Met Asn Ile Leu Leu Gln Tyr Val Va - #l Lys Ser Phe Asp Arg         #       125                                                                   - Ser Thr Lys Val Ile Asp Phe His Tyr Pro As - #n Glu Leu Leu Gln Glu         #   140                                                                       - Tyr Asn Trp Glu Leu Ala Asp Gln Pro Gln As - #n Leu Glu Glu Ile Leu         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Met His Cys Gln Thr Thr Leu Lys Tyr Ala Il - #e Lys Thr Gly His Pro         #               175                                                           - Arg Tyr Phe Asn Gln Leu Ser Thr Gly Leu As - #p Met Val Gly Leu Ala         #           190                                                               - Ala Asp Trp Leu Thr Ser Thr Ala Asn Thr As - #n Met Phe Thr Tyr Glu         #       205                                                                   - Ile Ala Pro Val Phe Val Leu Leu Glu Tyr Va - #l Thr Leu Lys Lys Met         #   220                                                                       - Arg Glu Ile Ile Gly Trp Pro Gly Gly Ser Gl - #y Asp Gly Ile Phe Ser         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Pro Gly Gly Ala Ile Ser Asn Met Tyr Ala Me - #t Met Ile Ala Arg Phe         #               255                                                           - Lys Met Phe Pro Glu Val Lys Glu Lys Gly Me - #t Ala Ala Leu Pro Arg         #           270                                                               - Leu Ile Ala Phe Thr Ser Glu His Ser His Ph - #e Ser Leu Lys Lys Gly         #       285                                                                   - Ala Ala Ala Leu Gly Ile Gly Thr Asp Ser Va - #l Ile Leu Ile Lys Cys         #   300                                                                       - Asp Glu Arg Gly Lys Met Ile Pro Ser Asp Le - #u Glu Arg Arg Ile Leu         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Glu Ala Lys Gln Lys Gly Phe Val Pro Phe Le - #u Val Ser Ala Thr Ala         #               335                                                           - Gly Thr Thr Val Tyr Gly Ala Phe Asp Pro Le - #u Leu Ala Val Ala Asp         #           350                                                               - Ile Cys Lys Lys Tyr Lys Ile Trp Met His Va - #l Asp Ala Ala Trp Gly         #       365                                                                   - Gly Gly Leu Leu Met Ser Arg Lys His Lys Tr - #p Lys Leu Ser Gly Val         #   380                                                                       - Glu Arg Ala Asn Ser Val Thr Trp Asn Pro Hi - #s Ile Met Met Gly Val         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Pro Leu Gln Cys Ser Ala Leu Leu Val Arg Gl - #u Glu Gly Leu Met Gln         #               415                                                           - Asn Cys Asn Gln Met His Ala Ser Tyr Leu Ph - #e Gln Gln Asp Lys His         #           430                                                               - Tyr Asp Leu Ser Tyr Asp Thr Gly Asp Lys Al - #a Leu Gln Cys Gly Arg         #       445                                                                   - His Val Asp Val Phe Lys Leu Trp Leu Met Tr - #p Arg Ala Lys Gly Thr         #   460                                                                       - Thr Gly Phe Glu Ala His Val Asp Lys Cys Le - #u Glu Leu Ala Glu Tyr         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Leu Tyr Asn Ile Ile Lys Asn Arg Glu Gly Ty - #r Glu Met Val Phe Asp         #               495                                                           - Gly Lys Pro Gln His Thr Asn Val Cys Phe Tr - #p Tyr Ile Pro Pro Ser         #           510                                                               - Leu Arg Thr Leu Glu Asp Asn Glu Glu Arg Me - #t Ser Arg Leu Ser Lys         #       525                                                                   - Val Ala Pro Val Ile Lys Ala Arg Met Met Gl - #u Tyr Gly Thr Thr Met         #   540                                                                       - Val Ser Tyr Gln Pro Leu Gly Asp Lys Val As - #n Phe Phe Arg Met Val         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Ile Ser Asn Pro Ala Ala Thr His Gln Asp Il - #e Asp Phe Leu Ile Glu         #               575                                                           - Glu Ile Glu Arg Leu Gly Gln Asp Leu                                         #           585                                                               - <210> SEQ ID NO 5                                                           <211> LENGTH: 585                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:ModifiedATION: Description of Artificial                                  hGAD                                                                    - <400> SEQUENCE: 5                                                           - Met Ala Ser Pro Gly Ser Gly Phe Trp Ser Ph - #e Gly Ser Glu Asp Gly         #                 15                                                          - Ser Gly Asp Ser Glu Asn Pro Gly Thr Ala Ar - #g Ala Trp Cys Gln Val         #             30                                                              - Ala Gln Lys Phe Thr Gly Gly Ile Gly Asn Ly - #s Leu Cys Ala Leu Leu         #         45                                                                  - Tyr Gly Asp Ala Glu Lys Pro Ala Glu Ser Gl - #y Gly Ser Gln Pro Pro         #     60                                                                      - Arg Ala Ala Ala Arg Lys Ala Ala Cys Ala Cy - #s Asp Gln Lys Pro Cys         # 80                                                                          - Ser Cys Ser Lys Val Asp Val Asn Tyr Ala Ph - #e Leu His Ala Thr Asp         #                 95                                                          - Leu Leu Pro Ala Cys Asp Gly Glu Arg Pro Th - #r Leu Ala Phe Leu Gln         #           110                                                               - Asp Val Met Asn Ile Leu Leu Gln Tyr Val Va - #l Lys Ser Phe Asp Arg         #       125                                                                   - Ser Thr Lys Val Ile Asp Phe His Tyr Pro As - #n Glu Leu Leu Gln Glu         #   140                                                                       - Tyr Asn Trp Glu Leu Ala Asp Gln Pro Gln As - #n Leu Glu Glu Ile Leu         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Met His Cys Gln Thr Thr Leu Lys Tyr Ala Il - #e Lys Thr Gly His Pro         #               175                                                           - Arg Tyr Phe Asn Gln Leu Ser Thr Gly Leu As - #p Met Val Gly Leu Ala         #           190                                                               - Ala Asp Trp Leu Thr Ser Thr Ala Asn Thr As - #n Met Phe Thr Tyr Glu         #       205                                                                   - Ile Ala Pro Val Phe Val Leu Leu Glu Tyr Va - #l Thr Leu Lys Lys Met         #   220                                                                       - Arg Glu Ile Ile Gly Trp Pro Gly Gly Ser Gl - #y Asp Gly Ile Phe Ser         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Pro Gly Gly Ala Ile Ser Asn Met Tyr Ala Me - #t Met Ile Ala Arg Phe         #               255                                                           - Lys Met Phe Pro Glu Val Lys Glu Lys Gly Me - #t Ala Ala Leu Pro Arg         #           270                                                               - Leu Ile Ala Phe Thr Ser Glu His Ser His Ph - #e Ser Leu Lys Lys Gly         #       285                                                                   - Ala Ala Ala Leu Gly Ile Gly Thr Asp Ser Va - #l Ile Leu Ile Lys Cys         #   300                                                                       - Asp Glu Arg Gly Lys Met Ile Pro Ser Asp Le - #u Glu Arg Arg Ile Leu         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Glu Ala Lys Gln Lys Gly Phe Val Pro Phe Le - #u Val Ser Ala Thr Ala         #               335                                                           - Gly Thr Thr Val Tyr Gly Ala Phe Asp Pro Le - #u Leu Ala Val Ala Asp         #           350                                                               - Ile Cys Lys Lys Tyr Lys Ile Trp Met His Va - #l Asp Ala Ala Trp Gly         #       365                                                                   - Gly Gly Leu Leu Met Ser Arg Lys His Lys Tr - #p Lys Leu Ser Gly Val         #   380                                                                       - Glu Arg Ala Asn Ser Val Thr Trp Asn Pro Hi - #s Arg Met Met Gly Val         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Pro Leu Gln Cys Ser Ala Leu Leu Val Arg Gl - #u Glu Gly Leu Met Gln         #               415                                                           - Asn Cys Asn Gln Met His Ala Ser Tyr Leu Ph - #e Gln Gln Asp Lys His         #           430                                                               - Tyr Asp Leu Ser Tyr Asp Thr Gly Asp Lys Al - #a Leu Gln Cys Gly Arg         #       445                                                                   - His Val Asp Val Phe Lys Leu Trp Leu Met Tr - #p Arg Ala Lys Gly Thr         #   460                                                                       - Thr Gly Phe Glu Ala His Val Asp Lys Cys Le - #u Glu Leu Ala Glu Tyr         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Leu Tyr Asn Ile Ile Lys Asn Arg Glu Gly Ty - #r Glu Met Val Phe Asp         #               495                                                           - Gly Lys Pro Gln His Thr Asn Val Cys Phe Tr - #p Tyr Ile Pro Pro Ser         #           510                                                               - Leu Arg Thr Leu Glu Asp Asn Glu Glu Arg Me - #t Ser Arg Leu Ser Lys         #       525                                                                   - Val Ala Pro Val Ile Lys Ala Arg Met Met Gl - #u Tyr Gly Thr Thr Met         #   540                                                                       - Val Ser Tyr Gln Pro Leu Gly Asp Lys Val As - #n Phe Phe Arg Met Val         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Ile Ser Asn Pro Ala Ala Thr His Gln Asp Il - #e Asp Phe Leu Ile Glu         #               575                                                           - Glu Ile Glu Arg Leu Gly Gln Asp Leu                                         #           585                                                               - <210> SEQ ID NO 6                                                           <211> LENGTH: 585                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:ModifiedATION: Description of Artificial                                  hGAD                                                                    - <400> SEQUENCE: 6                                                           - Met Ala Ser Pro Gly Ser Gly Phe Trp Ser Ph - #e Gly Ser Glu Asp Gly         #                 15                                                          - Ser Gly Asp Ser Glu Asn Pro Gly Thr Ala Ar - #g Ala Trp Cys Gln Val         #             30                                                              - Ala Gln Lys Phe Thr Gly Gly Ile Gly Asn Ly - #s Leu Cys Ala Leu Leu         #         45                                                                  - Tyr Gly Asp Ala Glu Lys Pro Ala Glu Ser Gl - #y Gly Ser Gln Pro Pro         #     60                                                                      - Arg Ala Ala Ala Arg Lys Ala Ala Cys Ala Cy - #s Asp Gln Lys Pro Cys         # 80                                                                          - Ser Cys Ser Lys Val Asp Val Asn Tyr Ala Ph - #e Leu His Ala Thr Asp         #                 95                                                          - Leu Leu Pro Ala Cys Asp Gly Glu Arg Pro Th - #r Leu Ala Phe Leu Gln         #           110                                                               - Asp Val Met Asn Ile Leu Leu Gln Tyr Val Va - #l Lys Ser Phe Asp Arg         #       125                                                                   - Ser Thr Lys Val Ile Asp Phe His Tyr Pro As - #n Glu Leu Leu Gln Glu         #   140                                                                       - Tyr Asn Trp Glu Leu Ala Asp Gln Pro Gln As - #n Leu Glu Glu Ile Leu         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Met His Cys Gln Thr Thr Leu Lys Tyr Ala Il - #e Lys Thr Gly His Pro         #               175                                                           - Arg Tyr Phe Asn Gln Leu Ser Thr Gly Leu As - #p Met Val Gly Leu Ala         #           190                                                               - Ala Asp Trp Leu Thr Ser Thr Ala Asn Thr As - #n Met Phe Thr Tyr Glu         #       205                                                                   - Ile Ala Pro Val Phe Val Leu Leu Glu Tyr Va - #l Thr Leu Lys Lys Met         #   220                                                                       - Arg Glu Ile Ile Gly Trp Pro Gly Gly Ser Gl - #y Asp Gly Ile Phe Ser         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Pro Gly Gly Ala Ile Ser Asn Met Tyr Ala Me - #t Met Ile Ala Arg Phe         #               255                                                           - Lys Met Phe Pro Glu Val Lys Glu Lys Gly Me - #t Ala Ala Leu Pro Arg         #           270                                                               - Leu Ile Ala Phe Thr Ser Glu His Ser His Ph - #e Ser Leu Lys Lys Gly         #       285                                                                   - Ala Ala Ala Leu Gly Ile Gly Thr Asp Ser Va - #l Ile Leu Ile Lys Cys         #   300                                                                       - Asp Glu Arg Gly Lys Met Ile Pro Ser Asp Le - #u Glu Arg Arg Ile Leu         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Glu Ala Lys Gln Lys Gly Phe Val Pro Phe Le - #u Val Ser Ala Thr Ala         #               335                                                           - Gly Thr Thr Val Tyr Gly Ala Phe Asp Pro Le - #u Leu Ala Val Ala Asp         #           350                                                               - Ile Cys Lys Lys Tyr Lys Ile Trp Met His Va - #l Asp Ala Ala Trp Gly         #       365                                                                   - Gly Gly Leu Leu Met Ser Arg Lys His Lys Tr - #p Lys Leu Ser Gly Val         #   380                                                                       - Glu Arg Ala Asn Ser Val Thr Trp Asn Pro Hi - #s Gln Met Met Gly Val         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Pro Leu Gln Cys Ser Ala Leu Leu Val Arg Gl - #u Glu Gly Leu Met Gln         #               415                                                           - Asn Cys Asn Gln Met His Ala Ser Tyr Leu Ph - #e Gln Gln Asp Lys His         #           430                                                               - Tyr Asp Leu Ser Tyr Asp Thr Gly Asp Lys Al - #a Leu Gln Cys Gly Arg         #       445                                                                   - His Val Asp Val Phe Lys Leu Trp Leu Met Tr - #p Arg Ala Lys Gly Thr         #   460                                                                       - Thr Gly Phe Glu Ala His Val Asp Lys Cys Le - #u Glu Leu Ala Glu Tyr         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Leu Tyr Asn Ile Ile Lys Asn Arg Glu Gly Ty - #r Glu Met Val Phe Asp         #               495                                                           - Gly Lys Pro Gln His Thr Asn Val Cys Phe Tr - #p Tyr Ile Pro Pro Ser         #           510                                                               - Leu Arg Thr Leu Glu Asp Asn Glu Glu Arg Me - #t Ser Arg Leu Ser Lys         #       525                                                                   - Val Ala Pro Val Ile Lys Ala Arg Met Met Gl - #u Tyr Gly Thr Thr Met         #   540                                                                       - Val Ser Tyr Gln Pro Leu Gly Asp Lys Val As - #n Phe Phe Arg Met Val         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Ile Ser Asn Pro Ala Ala Thr His Gln Asp Il - #e Asp Phe Leu Ile Glu         #               575                                                           - Glu Ile Glu Arg Leu Gly Gln Asp Leu                                         #           585                                                               - <210> SEQ ID NO 7                                                           <211> LENGTH: 585                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:ModifiedATION: Description of Artificial                                  hGAD                                                                    - <400> SEQUENCE: 7                                                           - Met Ala Ser Pro Gly Ser Gly Phe Trp Ser Ph - #e Gly Ser Glu Asp Gly         #                 15                                                          - Ser Gly Asp Ser Glu Asn Pro Gly Thr Ala Ar - #g Ala Trp Cys Gln Val         #             30                                                              - Ala Gln Lys Phe Thr Gly Gly Ile Gly Asn Ly - #s Leu Cys Ala Leu Leu         #         45                                                                  - Tyr Gly Asp Ala Glu Lys Pro Ala Glu Ser Gl - #y Gly Ser Gln Pro Pro         #     60                                                                      - Arg Ala Ala Ala Arg Lys Ala Ala Cys Ala Cy - #s Asp Gln Lys Pro Cys         # 80                                                                          - Ser Cys Ser Lys Val Asp Val Asn Tyr Ala Ph - #e Leu His Ala Thr Asp         #                 95                                                          - Leu Leu Pro Ala Cys Asp Gly Glu Arg Pro Th - #r Leu Ala Phe Leu Gln         #           110                                                               - Asp Val Met Asn Ile Leu Leu Gln Tyr Val Va - #l Lys Ser Phe Asp Arg         #       125                                                                   - Ser Thr Lys Val Ile Asp Phe His Tyr Pro As - #n Glu Leu Leu Gln Glu         #   140                                                                       - Tyr Asn Trp Glu Leu Ala Asp Gln Pro Gln As - #n Leu Glu Glu Ile Leu         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Met His Cys Gln Thr Thr Leu Lys Tyr Ala Il - #e Lys Thr Gly His Pro         #               175                                                           - Arg Tyr Phe Asn Gln Leu Ser Thr Gly Leu As - #p Met Val Gly Leu Ala         #           190                                                               - Ala Asp Trp Leu Thr Ser Thr Ala Asn Thr As - #n Met Phe Thr Tyr Glu         #       205                                                                   - Ile Ala Pro Val Phe Val Leu Leu Glu Tyr Va - #l Thr Leu Lys Lys Met         #   220                                                                       - Arg Glu Ile Ile Gly Trp Pro Gly Gly Ser Gl - #y Asp Gly Ile Phe Ser         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Pro Gly Gly Ala Ile Ser Asn Met Tyr Ala Me - #t Met Ile Ala Arg Phe         #               255                                                           - Lys Met Phe Pro Glu Val Lys Glu Lys Gly Me - #t Ala Ala Leu Pro Arg         #           270                                                               - Leu Ile Ala Phe Thr Ser Glu His Ser His Ph - #e Ser Leu Lys Lys Gly         #       285                                                                   - Ala Ala Ala Leu Gly Ile Gly Thr Asp Ser Va - #l Ile Leu Ile Lys Cys         #   300                                                                       - Asp Glu Arg Gly Lys Met Ile Pro Ser Asp Le - #u Glu Arg Arg Ile Leu         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Glu Ala Lys Gln Lys Gly Phe Val Pro Phe Le - #u Val Ser Ala Thr Ala         #               335                                                           - Gly Thr Thr Val Tyr Gly Ala Phe Asp Pro Le - #u Leu Ala Val Ala Asp         #           350                                                               - Ile Cys Lys Lys Tyr Lys Ile Trp Met His Va - #l Asp Ala Ala Trp Gly         #       365                                                                   - Gly Gly Leu Leu Met Ser Arg Lys His Lys Tr - #p Lys Leu Ser Gly Val         #   380                                                                       - Glu Arg Ala Asn Ser Val Thr Trp Asn Pro Hi - #s His Met Met Gly Val         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Pro Leu Gln Cys Ser Ala Leu Leu Val Arg Gl - #u Glu Gly Leu Met Gln         #               415                                                           - Asn Cys Asn Gln Met His Ala Ser Tyr Leu Ph - #e Gln Gln Asp Lys His         #           430                                                               - Tyr Asp Leu Ser Tyr Asp Thr Gly Asp Lys Al - #a Leu Gln Cys Gly Arg         #       445                                                                   - His Val Asp Val Phe Lys Leu Trp Leu Met Tr - #p Arg Ala Lys Gly Thr         #   460                                                                       - Thr Gly Phe Glu Ala His Val Asp Lys Cys Le - #u Glu Leu Ala Glu Tyr         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Leu Tyr Asn Ile Ile Lys Asn Arg Glu Gly Ty - #r Glu Met Val Phe Asp         #               495                                                           - Gly Lys Pro Gln His Thr Asn Val Cys Phe Tr - #p Tyr Ile Pro Pro Ser         #           510                                                               - Leu Arg Thr Leu Glu Asp Asn Glu Glu Arg Me - #t Ser Arg Leu Ser Lys         #       525                                                                   - Val Ala Pro Val Ile Lys Ala Arg Met Met Gl - #u Tyr Gly Thr Thr Met         #   540                                                                       - Val Ser Tyr Gln Pro Leu Gly Asp Lys Val As - #n Phe Phe Arg Met Val         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Ile Ser Asn Pro Ala Ala Thr His Gln Asp Il - #e Asp Phe Leu Ile Glu         #               575                                                           - Glu Ile Glu Arg Leu Gly Gln Asp Leu                                         #           585                                                               - <210> SEQ ID NO 8                                                           <211> LENGTH: 585                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:ModifiedATION: Description of Artificial                                  hGAD                                                                    - <400> SEQUENCE: 8                                                           - Met Ala Ser Pro Gly Ser Gly Phe Trp Ser Ph - #e Gly Ser Glu Asp Gly         #                 15                                                          - Ser Gly Asp Ser Glu Asn Pro Gly Thr Ala Ar - #g Ala Trp Cys Gln Val         #             30                                                              - Ala Gln Lys Phe Thr Gly Gly Ile Gly Asn Ly - #s Leu Cys Ala Leu Leu         #         45                                                                  - Tyr Gly Asp Ala Glu Lys Pro Ala Glu Ser Gl - #y Gly Ser Gln Pro Pro         #     60                                                                      - Arg Ala Ala Ala Arg Lys Ala Ala Cys Ala Cy - #s Asp Gln Lys Pro Cys         # 80                                                                          - Ser Cys Ser Lys Val Asp Val Asn Tyr Ala Ph - #e Leu His Ala Thr Asp         #                 95                                                          - Leu Leu Pro Ala Cys Asp Gly Glu Arg Pro Th - #r Leu Ala Phe Leu Gln         #           110                                                               - Asp Val Met Asn Ile Leu Leu Gln Tyr Val Va - #l Lys Ser Phe Asp Arg         #       125                                                                   - Ser Thr Lys Val Ile Asp Phe His Tyr Pro As - #n Glu Leu Leu Gln Glu         #   140                                                                       - Tyr Asn Trp Glu Leu Ala Asp Gln Pro Gln As - #n Leu Glu Glu Ile Leu         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Met His Cys Gln Thr Thr Leu Lys Tyr Ala Il - #e Lys Thr Gly His Pro         #               175                                                           - Arg Tyr Phe Asn Gln Leu Ser Thr Gly Leu As - #p Met Val Gly Leu Ala         #           190                                                               - Ala Asp Trp Leu Thr Ser Thr Ala Asn Thr As - #n Met Phe Thr Tyr Glu         #       205                                                                   - Ile Ala Pro Val Phe Val Leu Leu Glu Tyr Va - #l Thr Leu Lys Lys Met         #   220                                                                       - Arg Glu Ile Ile Gly Trp Pro Gly Gly Ser Gl - #y Asp Gly Ile Phe Ser         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Pro Gly Gly Ala Ile Ser Asn Met Tyr Ala Me - #t Met Ile Ala Arg Phe         #               255                                                           - Lys Met Phe Pro Glu Val Lys Glu Lys Gly Me - #t Ala Ala Leu Pro Arg         #           270                                                               - Leu Ile Ala Phe Thr Ser Glu His Ser His Ph - #e Ser Leu Lys Lys Gly         #       285                                                                   - Ala Ala Ala Leu Gly Ile Gly Thr Asp Ser Va - #l Ile Leu Ile Lys Cys         #   300                                                                       - Asp Glu Arg Gly Lys Met Ile Pro Ser Asp Le - #u Glu Arg Arg Ile Leu         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Glu Ala Lys Gln Lys Gly Phe Val Pro Phe Le - #u Val Ser Ala Thr Ala         #               335                                                           - Gly Thr Thr Val Tyr Gly Ala Phe Asp Pro Le - #u Leu Ala Val Ala Asp         #           350                                                               - Ile Cys Lys Lys Tyr Lys Ile Trp Met His Va - #l Asp Ala Ala Trp Gly         #       365                                                                   - Gly Gly Leu Leu Met Ser Arg Lys His Lys Tr - #p Lys Leu Ser Gly Val         #   380                                                                       - Glu Arg Ala Asn Ser Val Thr Trp Asn Pro Hi - #s Gly Met Met Gly Val         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Pro Leu Gln Cys Ser Ala Leu Leu Val Arg Gl - #u Glu Gly Leu Met Gln         #               415                                                           - Asn Cys Asn Gln Met His Ala Ser Tyr Leu Ph - #e Gln Gln Asp Lys His         #           430                                                               - Tyr Asp Leu Ser Tyr Asp Thr Gly Asp Lys Al - #a Leu Gln Cys Gly Arg         #       445                                                                   - His Val Asp Val Phe Lys Leu Trp Leu Met Tr - #p Arg Ala Lys Gly Thr         #   460                                                                       - Thr Gly Phe Glu Ala His Val Asp Lys Cys Le - #u Glu Leu Ala Glu Tyr         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Leu Tyr Asn Ile Ile Lys Asn Arg Glu Gly Ty - #r Glu Met Val Phe Asp         #               495                                                           - Gly Lys Pro Gln His Thr Asn Val Cys Phe Tr - #p Tyr Ile Pro Pro Ser         #           510                                                               - Leu Arg Thr Leu Glu Asp Asn Glu Glu Arg Me - #t Ser Arg Leu Ser Lys         #       525                                                                   - Val Ala Pro Val Ile Lys Ala Arg Met Met Gl - #u Tyr Gly Thr Thr Met         #   540                                                                       - Val Ser Tyr Gln Pro Leu Gly Asp Lys Val As - #n Phe Phe Arg Met Val         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Ile Ser Asn Pro Ala Ala Thr His Gln Asp Il - #e Asp Phe Leu Ile Glu         #               575                                                           - Glu Ile Glu Arg Leu Gly Gln Asp Leu                                         #           585                                                               - <210> SEQ ID NO 9                                                           <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Homo sapiens                                                  <220> FEATURE:                                                                #Sequence:PLP bindingON: Description of Artificial                                  site of hGAD65                                                          - <400> SEQUENCE: 9                                                           - Thr Trp Asn Pro His Lys Met Met Gly Val                                     #                 10                                                          - <210> SEQ ID NO 10                                                          <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:Mutant PLPION: Description of Artificial                                  binding site of hGAD65                                                  - <400> SEQUENCE: 10                                                          - Thr Trp Asn Pro His Arg Met Met Gly Val                                     #                 10                                                          - <210> SEQ ID NO 11                                                          <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:Mutant PLPION: Description of Artificial                                  binding site of hGAD65                                                  - <400> SEQUENCE: 11                                                          - Thr Trp Asn Pro His Ile Met Met Gly Val                                     #                 10                                                          - <210> SEQ ID NO 12                                                          <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:Mutant PLPION: Description of Artificial                                  binding site of hGAD65                                                  - <400> SEQUENCE: 12                                                          - Thr Trp Asn Pro His Gln Met Met Gly Val                                     #                 10                                                          - <210> SEQ ID NO 13                                                          <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:Mutant PLPION: Description of Artificial                                  binding site of hGAD65                                                  - <400> SEQUENCE: 13                                                          - Thr Trp Asn Pro His His Met Met Gly Val                                     #                 10                                                          - <210> SEQ ID NO 14                                                          <211> LENGTH: 10                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:Mutant PLPION: Description of Artificial                                  binding site of hGAD65                                                  - <400> SEQUENCE: 14                                                          - Thr Trp Asn Pro His Gly Met Met Gly Val                                     #                 10                                                          __________________________________________________________________________

What is claimed is:
 1. A modified glutamic acid decarboxylase comprisingamino acid sequence (SEQ ID NO:3):

    Met Ala Ser Pro Gly Ser Gly Phe Trp Ser Phe Gly Ser                             1               5                  10                                       Glu Asp Gly Ser Gly Asp Ser Glu Asn Pro Gly Thr Ala                                15                  20                  25                               Arg Ala Trp Cys Gln Val Ala Gln Lys Phe Thr Gly Gly                                        30                  35                                           Ile Gly Asn Lys Leu Cys Ala Leu Leu Tyr Gly Asp Ala                            40                  45                  50                                   Glu Lys Pro Ala Glu Ser Gly Gly Ser Gln Pro Pro Arg                                    55                  60                  65                           Ala Ala Ala Arg Lys Ala Ala Cys Ala Cys Asp Gln Lys                                            70                  75                                       Pro Cys Ser Cys Ser Lys Val Asp Val Asn Tyr Ala Phe                                80                  85                  90                               Leu His Ala Thr Asp Leu Leu Pro Ala Cys Asp Gly Glu                                        95                 100                                           Arg Pro Thr Leu Ala Phe Leu Gln Asp Val Met Asn Ile                           105                 110                 115                                   Leu Leu Gln Tyr Val Val Lys Ser Phe Asp Arg Ser Thr                                   120                 125                 130                           Lys Val Ile Asp Phe His Tyr Pro Asn Glu Leu Leu Gln                                           135                 140                                       Glu Tyr Asn Trp Glu Leu Ala Asp Gln Pro Gln Asn Leu                           145                 150                 155                                   Glu Glu Ile Leu Met His Cys Gln Thr Thr Leu Lys Tyr                                       160                 165                                           Ala Ile Lys Thr Gly His Pro Arg Tyr Phe Asn Gln Leu                           170                 175                 180                                   Ser Thr Gly Leu Asp Met Val Gly Leu Ala Ala Asp Trp                                   185                 190                 195                           Leu Thr Ser Thr Ala Asn Thr Asn Met Phe Thr Tyr Glu                                           200                 205                                       Ile Ala Pro Val Phe Val Leu Leu Glu Tyr Val Thr Leu                               210                 215                 220                               Lys Lys Met Arg Glu Ile Ile Gly Trp Pro Gly Gly Ser                                       225                 230                                           Gly Asp Gly Ile Phe Ser Pro Gly Gly Ala Ile Ser Asn                           235                 240                 245                                   Met Tyr Ala Met Met Ile Ala Arg Phe Lys Met Phe Pro                                   250                 255                 260                           Glu Val Lys Glu Lys Gly Met Ala Ala Leu Pro Arg Leu                                           265                 270                                       Ile Ala Phe Thr Ser Glu His Ser His Phe Ser Leu Lys                               275                 280                 285                               Lys Gly Ala Ala Ala Leu Gly Ile Gly Thr Asp Ser Val                                       290                 295                                           Ile Leu Ile Lys Cys Asp Glu Arg Gly Lys Met Ile Pro                           300                 305                 310                                   Ser Asp Leu Glu Arg Arg Ile Leu Glu Ala Lys Gln Lys                                   315                 320                 325                           Gly Phe Val Pro Phe Leu Val Ser Ala Thr Ala Gly Thr                                           330                 335                                       Thr Val Tyr Gly Ala Phe Asp Pro Leu Leu Ala Val Ala                               340                 345                 350                               Asp Ile Cys Lys Lys Tyr Lys Ile Trp Met His Val Asp                                       355                 360                                           Ala Ala Trp Gly Gly Gly Leu Leu Met Ser Arg Lys His                           365                 370                 375                                   Lys Trp Lys Leu Ser Gly Val Glu Arg Ala Asn Ser Val                                   380                 385                 390                           Thr Trp Asn Pro His Xaa Met Met Gly Val Pro Leu Gln                                            395            400                                           Cys Ser Ala Leu Leu Val Arg Glu Glu Gly Leu Met Gln                               405                 410                 415                               Asn Cys Asn Gln Met His Ala Ser Tyr Leu Phe Gln Gln                                       420                 425                                           Asp Lys His Tyr Asp Leu Ser Tyr Asp Thr Gly Asp Lys                           430                 435                 440                                   Ala Leu Gln Cys Gly Arg His Val Asp Val Phe Lys Leu                                   445                 450                 455                           Trp Leu Met Trp Arg Ala Lys Gly Thr Thr Gly Phe Glu                                           460                 465                                       Ala His Val Asp Lys Cys Leu Glu Leu Ala Glu Tyr Leu                               470                 475                 480                               Tyr Asn Ilr Ile Lys Asn Arg Glu Gly Tyr Glu Met Val                                       485                 490                                           Phe Asp Gly Lys Pro Gln His Thr Asn Val Cys Phe Trp                           495                 500                 505                                   Tyr Ile Pro Pro Ser Leu Arg Thr Leu Glu Asp Asn Glu                                   510                 515                 520                           Glu Arg Met Ser Arg Leu Ser Lys Val Ala Pro Val Ile                                           525                 530                                       Lys Ala Arg Met Met Glu Tyr Gly Thr Thr Met Val Ser                               535                 540                 545                               Tyr Gln Pro Leu Gly Asp Lys Val Asn Phe Phe Arg Met                                       550                 555                                           Val Ile Ser Asn Pro Ala Ala Thr His Gln Asp Ile Asp                           560                 565                 570                                   Phe Leu Ile Glu Glu Ile Glu Arg Leu Gly Gln Asp Leu                                   575                 580                 585                       

wherein Xaa represents a L amino acid chosen from the group ofisoleucine (SEQ ID NO:4), arginine (SEQ ID NO:5), glutamine (SEQ IDNO:6), histidine (SEQ ID NO:7) and glycine (SEQ ID NO:8).
 2. A modifiedglutamic acid decarboxylase according to claim 1, wherein Xaa isarginine (SEQ ID NO:5).
 3. A nucleotide sequence encoding a modifiedglutamic acid decarboxylase according to claim
 1. 4. A nucleotidesequence according to claim 3 which is the one disclosed herein as SEQID NO:2.
 5. A vector containing the nucleic acid sequence according toclaim
 3. 6. A host cell containing a vector according to claim
 5. 7. Aprocedure for producing a modified glutamic acid decarboxylasecomprising cultivating a host cell according to claim
 6. 8. Apharmaceutical composition comprising a therapeutically effective amountof a modified glutamic acid decarboxylase according to claim 1 togetherwith a pharmaceutically acceptable excipient.
 9. A method for treatingand/or preventing autoimmune disorders, comprising administering to ahuman or animal in need thereof, an effective amount of a modifiedglutamic acid decarboxylase according to claim 1, said amount beingeffective to treat and/or prevent said autoimmune disorder.
 10. Anoligonucleotide encoding a modified portion of a modified glutamic aciddecarboxylase, wherein said modified portion encodes the amino acidsequence consisting of amino acids 391-400 of SEQ ID NO:3, wherein theamino acid at postion 396 of SEQ ID NO:3 is replaced with an amino acidselected from the group consisting of isoleucine (SEQ ID NO:11),arginine (SEQ ID NO:10), glutamine (SEQ ID NO:12), histidine (SEQ IDNO:13) and glycine (SEQ ID NO:14).
 11. An oligonucleotide according toclaim 10 comprising the sequence disclosed as SEQ ID NO:1.